We are interested in the characterization of two proteins of Rickettsia rickettsii, the etiological agent of Rocky Mountain spotted fever (RMSF). These two proteins (120 and 155 kDa) are immunogenic, have been implicated as virulence factors, and may be subunit vaccine candidates. We cloned the gene encoding the 155- kDa protein and showed that immunization with Escherichia coli producing this protein protected guinea pigs challenged with viable R. rickettsii 7 days postvaccination. These studies were extended to determine whether boosting the animals 7 days after the first immunization would enhance the protection. The gene encoding a 197-kDa protein in R. conorii, the cause of Boutonneuse fever, is also being cloned; this protein is of interest because it is immunologically related to the 155-kDa protein of R. Rickettsii. We also cloned the R. rickettsii gene encoding the 12-kDa and successfully subcloned a portion of the gene into bacteriophage lambda-gt11. E. coli strain Y1090, made lysogenic for the recombinant bacteriophage (lambda-NEJ120), produced a 130-kDa protein that reacted by immunoblotting with a monospecific polyclonal serum raised to the 120-kDa rickettsial protein and beta-galactosidase. The 3.7-kilobase (kb) insert of lambda-NEJ120 was subcloned into the plasmid vector pUC19 to yield pGAM120, portions of which were subcloned into M13 vectors for DNA sequencing. The pGAM120 insert was used as a probe to screen a R. rickettsii (R strain) genomic library made in lambda-EMBL3 and a R. rickettsii (HLP strain) library made in pBR322; the HLP strain is avirulent in guinea pigs and is also thought to be avirulent in humans. Positive recombinants were obtained from both libraries. We have also continued our efforts to evaluate the use of monoclonal antibodies to therapeutically treat RMSF.